Multi-sample fermentor and method of using same

ABSTRACT

A fermentation apparatus is constructed to produce a known and repeatable amount of untainted fermentation product using multiple fermentation vessels. To facilitate further processing compatible with other product processing steps, the fermentation apparatus has an array of sample vessels arranged in a container frame. The container frame is configured to hold the sample vessels during fermentation and to transport the vessel array to or from another processing station. Corresponding to the number of sample vessels in the sample vessel array, a cannula array is configured such that each cannula may be placed inside a sample vessel. The cannula array is attached to a gas distributor that delivers oxygen and/or one or more other gases from a gas source through the cannula into the sample vessel. Because the fermentation volume for each individual sample vessel is smaller than a bulk fermentation apparatus, the fermentation product yields are predictable and cell growth rates can be effectively optimized.

COPYRIGHT NOTIFICATION

[0001] Pursuant to 37 C.F.R. 1.71(e), a portion of this patent documentcontains material which is subject to copyright protection. Thecopyright owner has no objection to the facsimile reproduction by anyoneof the patent document or the patent disclosure, as it appears in thePatent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] Pursuant to 35 U.S.C. § 120, and any other applicable statute orrule, the present application is a continuation-in-part of and claimsbenefit of and priority to U.S. patent application Ser. No. 09/780,591,filed Feb. 8, 2001 entitled “Multi-Sample Fermentor and Method of UsingSame,” the disclosure of which is incorporated herein by reference inits entirety for all purposes.

BACKGROUND OF THE INVENTION

[0003] Fermentation is a key technology in many fields and industriesand is performed both on a mass production scale and on an experimental,bench top scale. For example, fermentation systems are used for theproduction of a large number of products such as antibiotics, vaccines,synthetic biopolymers, synthetic amino acids, and proteins. Fermentationtechnology is integral in the production of recombinant proteins usingbiological organisms, such as E. coli, and many other cell cultures. Forexample, production of commercial pharmaceuticals such as recombinantinsulin (Eli Lilly), erythropoietin (Amgen), and interferon (Roche) allinvolve fermentation as an essential step.

[0004] In addition, the recent identification of the tens of thousandsof genes comprising the human genome highlight an important use offermentation, namely the production of the proteins encoded by thosegenes. The determination of each gene's function is of paramountimportance and therefore, the proteins encoded by those genes must beproduced, e.g., by fermentation methods. Because each gene encodes atleast one protein, tens of thousands of proteins must be produced andisolated. However, fermentation and isolation of the resulting proteinproducts typcially requires several labor intensive and time-consumingprocedures. Fermentation systems that can produce tens of thousands ofdifferent proteins, e.g., in amounts sufficient for analysis aretherefore needed. An additional advantage would be fermentation systemsthat are amenable to high throughput processes and the microtiter plateformat used in many biotechnolgy applications.

[0005] Although, rapid advances in biotechnology have enabled thedevelopment of high throughput alternatives to traditional laboratorybench top processes, fermentation methods have not been amenable toautomation. For example, limits in current fermentation technologyprevent the uninterrupted processing flow that characterizes automatedhigh throughput systems. Existing fermentation systems typically involvemultiple handling steps by either a batch processing method or acontinuous processing method.

[0006] Fermentations are typically carried out in batch mode orcontinuous mode. Batch mode processes are those in which a fermentor isfilled with a medium in which cells are grown and the fermentation isallowed to proceed with the entire contents removed from the fermentorat the end for downstream or post-processing. The fermentor is thencleaned, re-filled, and inoculated for the fermentation process to beperformed again. For example, current production scale batch processesinvolve first fermenting in large scale, bulk fermentation vessels, thenprocessing the fermentation medium to isolate the desired fermentationproduct, followed by transferring this product into the productionstream for further processing, and finally cleaning the fermentationapparatus for the next batch. In a large scale batch culture, it isgenerally necessary to provide a high initial concentration of nutrientsin order to sustain cell growth over an extended time. As a result,substrate inhibition may occur in the early stages of cell growth andthen may be followed by a nutrient deficiency in the late stages offermentation. These disadvantages result in sub-optimal cell growthrates and fermentation yields. Another disadvantage of this method liesin the need to individually dispense the fermentation products from thebulk fermentation apparatus into separate sample vessels for furtherprocessing. Thus, by producing the fermentation product on a bulk scale,the fermentation product is not immediately available for automatedprocessing. Further disadvantages include the decreased efficiency ofboth transferring the material to another sample vessel, as well ascleaning and sterilizing the fermentation apparatus for the next batch.These disadvantages result in increased production costs, inefficientproduction times and decreased yields.

[0007] Continuous batch processes involve siphoning off the fermentationproduct from the bulk fermentation vessel and continuously addingnutrients to the fermentation medium according to a calculatedexponential growth curve. This curve, however, is merely anapproximation that does not accurately predict cell growth in large,industrial scale quantities of fermentation medium. Consequently, due tothe unpredictable nature of large scale fermentation environments,experienced personnel are required to monitor the feeding rate veryclosely. Changes in the fermentation environment may result in eitherpoisoned fermentation products being siphoned off into the productionstream or sub-optimal production yields due to starved fermentationmediums. As a further disadvantage, unpredictable fermentation productyields affect the accuracy of subsequent processing steps. For example,when the fermentation yield decreases, the amount of aspirating, theamount of reagent dispensed, or the centrifuge time is no longeroptimized, or even predictable. Frequent or continuous monitoring of thefermentation process and adjustment of the fermentation conditions isoften not practicable or efficient in a production scale process.

[0008] Neither of the current processes provides an efficient, automatedproduction scale fermentation. However, fermentation remains a keyprocessing step in a number of industries, particularly in biotechnologyindustries, and thus a need exists for incorporating fermentationprocesses into automated high throughput systems. A process thatproduces a precise, known, and repeatable amount of untaintedfermentation product with limited human interaction or sample vesseltransfer is essential to integrating fermentation into modern productionprocesses. The present invention meets these as well as other needs thatwill be apparent upon review of the following detailed description andfigures.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods and apparatuses forsimultaneously fermenting a plurality of samples, e.g., small samples inan 8 by 12 array. For example, the present invention provides afermentation apparatus comprising a container frame configured tocontain a plurality of sample vessels and a gas distribution arrangementcoupled to the container frame. The fermentor provides for fermentationof large numbers of samples, e.g., to produce a large number ofproteins. Alternatively, the fermentors of the invention provide a moreefficient route for production scale fermentations.

[0010] In one aspect the invention provides a container frame configuredto contain a plurality of sample vessels, e.g., in an array; and, a gasdistribution arrangement configured to provide gas to a plurality ofsample vessels, e.g., when the sample vessels are positioned in thecontainer frame. The container frame is typically configured to containan array of sample vessels, e.g., an 8 by 12 array, e.g., holding atleast about 96, 384, or 1536 samples. The gas distribution arrangementtypically comprises a gas inlet configured to deliver gas to a pluralityof cannulas, which are configured to provide gas to the sample vessels.

[0011] In one embodiment, the container frame is a transportablecontainer frame, e.g., configured for transport to a post-fermentationprocessing station. In addition, the container frame is optionallyconfigured for placement within a temperature controlled area, e.g.,water bath or a temperature controlled room, wherein a temperaturecontroller is coupled to the container frame and/or to one or moresample vessels within the container frame.

[0012] In other embodiments, the container frame is autoclavable. Forexample, the container frame is autoclavable on its own or incombination with the gas distribution arrangement and/or the samplevessels.

[0013] The sample vessels typically comprise glass, plastic, metal,polycarbonate, ceramic, or the like. Each sample vessel typically has avolume of about 50 to 100 ml, e.g., and is used to hold a samplecomprising less than about 80 mls, more typically about 65 mls. Thesamples in the plurality of sample vessels each have substantially thesame composition or different compositions, e.g., to produce a largequantity of a single protein, or to produce multiple proteinssimultaneously.

[0014] In other embodiments, the sample vessels optionally comprise avent, e.g., for releasing built up pressure during fermentation. Sensorsare also optionally placed in contact with one or more of the samples inthe sample vessels, e.g., for monitoring temperature, pH, and the like.

[0015] In one embodiment, the gas distribution arrangement comprises adispensing plate, an array of sample vessel areas, an array of cannulas,and a gas inlet. The dispensing plate typically comprises a top portionand a bottom portion that are joined together such that a hollow spaceexists between them. The array of sample vessel areas is typicallylocated in a bottom surface of the bottom portion. Each sample vesselarea comprises a recess and is positioned to correspond to the array ofsample vessels. The array of cannulas are typically in fluidcommunication with the hollow space and protrude from a bottom surfaceof the dispensing plate through the sample vessel areas, e.g., toprovide gas flow to the sample vessels. In some embodiments, thecannulas comprises a plurality of passages, e.g., at least threepassages. The gas inlet is typically in fluid communication with thehollow space for delivering gas into a plurality of sample vessels viathe cannulas during fermentation. For example, the gas distributionarrangement in some embodiments comprises a gas source that providesoxygen or a mixture of oxygen and at least one other gas to each samplevessel during operation of the apparatus.

[0016] In other embodiments, the gas distribution arrangement isoptionally configured to allow delivery of one or more reagents to thesample vessels. For example, a dispenser is optionally coupled to thegas distribution arrangement, e.g., for dispensing one or more reagentsinto the plurality of sample vessels. The dispenser is typicallyconfigured to dispense reagents into the plurality of sample vessels,e.g., via a plurality of apertures corresponding to the sample vessels.

[0017] In addition, a process controller is operably coupled to the gasdistribution arrangement, e.g., for controlling and/or monitoring gasflow to the plurality of sample being fermented.

[0018] In another aspect, the present invention provides a fermentorhead for multiple sample fermentation. A typical fermentor headcomprises a dispensing plate that comprises a top portion and a bottomportion, an array of sample vessels areas, an array of cannulas and agas inlet. The bottom portion and the top portion of the dispensingplate are joined together such that a hollow space exists between them.The array of sample vessel areas is typically located in a bottomsurface of the bottom portion of the dispensing plate, which samplevessel areas each comprise a recess and are positioned to correspond toan array of sample vessels. The array of cannulas are typically in fluidcommunication with the hollow space and protrude from a bottom surfaceof the dispensing plate through the sample vessel areas, e.g., 15 to 16cm; with the gas inlet in fluid communication with the hollow space fordelivering gas into a plurality of sample vessels via the cannulasduring fermentation. Typically, the cannulas deliver gas adjacent to abottom of the sample vessels. In some embodiments, the dispensing platefurther comprises an array of apertures for accessing samples duringfermentation. Alternatively, the cannulas are adapted to deliver gas,deliver fluid, or aspirate fluid from the sample vessels duringfermentation. The vessels and samples used with the fermentor headtypically correspond to those described above.

[0019] In another aspect, the present invention provides a method offermenting a plurality of samples. The method typically comprisesproviding a plurality of sample vessels in a container frame, whereineach of the sample vessels contains a sample. The samples in theplurality of sample vessels are typically fermented, which fermentingcomprises simultaneously delivering gas, e.g., oxygen, air, and/or,nitrogen, to each of the sample vessels via a plurality of cannulasassociated with the sample vessels. Each sample typically has a volumeof less than 100 ml, e.g., using sample vessels and a container frame asdescribed above. In some embodiments, delivering gas to the samplescomprises delivering air and oxygen to the samples over a period oftime, during which period of time, the ratio of air to oxygen changes,e.g., linearly over time or in a stepwise manner over time.

[0020] In some embodiments, the methods further comprise detecting oneor more fermentation conditions with a sensor coupled to one or moresample vessels and adjusting the fermentation conditions in the samplevessels, e.g., at pre-determined time intervals. For example, adjustingthe fermentation conditions optionally comprises adding a feed solutionto the sample vessels. Detecting optionally comprises measuring a pH ofone of the samples; measuring a redox potential of one of the samples;measuring an optical density of one of the samples; and/or measuring alight emission from one of the samples.

[0021] In some embodiments, the methods further comprise pre-processingor post-processing the samples in the same set of sample vessels, e.g.,in the same or a different location as the fermentation step. In someembodiments, the pre-processing and/or post-processing are performedrobotically. Pre-processing and/or post-processing steps include, butare not limited to, centrifugation, aspiration, and/or dispensing of oneor more reagent. For example, the methods optionally comprisetransferring the sample vessels into a centrifuge rotor afterfermentation or autoclaving the sample vessels, e.g., in the containerframe prior to fermentation. In addition, the cannulas are alsooptionally autoclavable with the container frame.

[0022] In another aspect, the methods of the invention comprisepositioning a plurality of sample vessels into a transportable containerframe, which container frame maintains the sample vessels in an array.The plurality of samples is optionally placed into the plurality ofsample vessels, e.g., before or after the vessels are positioned in theframe. A fermentor head is typically attached to the container frame,e.g., prior to or after the samples have been added to the vessels. Thefermentor head typically comprises an array of cannulas, e.g., asdescribed above. The cannulas typically correspond to the array ofsample vessels and are inserted into the sample vessels when thefermentor head is attached. The samples in the sample vessels are thenfermented, e.g., by simultaneously delivering a gas, e.g., oxygen,nitrogen, and/or air, to the samples via the array of cannulas. In someembodiments, the fermentation is an anaerobic fermentation comprisingdelivering an inert gas to maintain anaerobic fermentation conditions inthe sample vessels. The methods optionally comprise robotic stepspre-processing steps, and/or post processing steps as described above.For example, the sample vessels and/or the sample container used in theabove methods are optionally configured to be compatible with acentrifuge, wherein the method further comprises transporting thecontainer frame and/or sample vessels to the centrifuge forcentrifugation.

[0023] In some embodiments, the methods comprise transportation to anaspirator or dispenser, wherein the aspirator typically comprises anaspirator head which corresponds to the array of sample vessels withinthe container frame, in which case, the method further includingoperably attaching the aspirator head to the sample vessels andsimultaneously aspirating the samples within the sample vessels. Inother embodiments, a dispensing step is included, wherein the dispensercomprises a dispensing head corresponding to the array of sample vesselsand the method further includes operably attaching the dispenser head tothe sample vessels and simultaneously dispensing one or more materialsinto the sample vessels.

[0024] In another embodiment, the present invention provides a method ofprocessing a plurality of fermentation samples. The method comprisesfermenting a plurality of fermentation samples in a plurality of samplevessels, resulting in a plurality of fermented samples; roboticallytransporting the sample vessels containing the fermented samples to acentrifuge head; and centrifuging the fermented samples in the samesample vessels in which the fermentation was performed. For example,about 4 to about 10 sample vessels are optionally roboticallytransported to the centrifuge head at the same time. The method alsooptionally includes isolating a supernatant from the sample vesselsafter centrifuging the fermentation samples.

BRIEF DESCRIPTION OF THE FIGURES

[0025]FIG. 1 is a schematic showing a perspective view of a fermentationapparatus in accordance with the present invention.

[0026]FIG. 2 is a schematic showing a top view of a fermentationapparatus in accordance with the present invention.

[0027]FIG. 3 is a schematic illustrating a perspective view of anindividual fermentation sample vessel in accordance with the presentinvention.

[0028]FIG. 4 is a block diagram of a fermentation method in accordancewith the present invention.

[0029]FIG. 5 is a block diagram showing the use of a fermentation systemwithin a multiple process procedure in accordance with the presentinvention.

[0030]FIG. 6 is a schematic illustrating a bottom view of a gasarrangement in accordance with the present invention.

[0031]FIG. 7 is an automated fermentation assembly in accordance withthe present invention.

[0032]FIG. 8 is a cross sectional view of a cannula in accordance withthe present invention.

[0033]FIG. 9 is schematic showing a bottom view of a sample vessel areaof a dispensing plate shown in FIG. 6 in accordance with the presentinvention.

[0034]FIG. 10 is a schematic showing a cross sectional view of thesample vessel area shown in FIG. 9 taken along the line E-E inaccordance with the present invention.

[0035]FIG. 11 is a schematic showing a cross sectional view of thesample vessel area shown in FIG. 9 taken along line F-F in accordancewith the present invention.

[0036]FIG. 12 is a schematic showing a perspective view of afermentation sample vessel employing a dispensing plate in accordancewith the present invention.

[0037]FIG. 13 is a schematic drawing that illustrates a container framefor maintaining a plurality of sample vessels in an array configuration.

[0038]FIG. 14 is a schematic drawing that illustrates the containerframe of FIG. 13 coupled to a gas distribution arrangement.

[0039]FIG. 15 is a schematic drawing that illustrates the containerframe of FIG. 13 coupled to an alternative gas distribution arrangementconfigured for liquid additions.

[0040]FIG. 16 is a schematic drawing that illustrates the gasdistribution manifold with a liquid addition capacity of FIG. 15.

[0041]FIG. 17 is a schematic drawing that illustrating a cross-sectionalview taken along line A-A of FIG. 16.

[0042]FIG. 18 is a schematic drawing that illustrates a bottom view ofgas distribution arrangement as shown in FIG. 14.

[0043]FIG. 19 is a detail illustration from FIG. 18.

[0044]FIG. 20 is a schematic drawing illustrating a cross-sectional viewof a gas distribution arrangement including top and bottom plates takenalong line B-B of FIG. 19.

[0045]FIG. 21 is a schematic drawing that provides a side view of thegas distribution arrangement as shown in FIG. 14.

DETAILED DISCUSSION OF THE INVENTION

[0046] The present invention provides a fermentation apparatus andmethods of fermentation. The fermentors and methods presented hereinprovide production scale fermentation, e.g., automated high throughputfermentation, as described below. For example, the present inventionprovides a multi-sample fermentor comprising a transportable containerframe. The fermentor is configured to simultaneously ferment a pluralityof samples held in an array of sample vessels within a container frame.The sample vessels provide relatively small volume batch fermentations,e.g., about 50 ml to 80 ml of sample in each sample vessel, moretypically 65 ml. In addition, the transportable container frame providesa high throughput aspect to the system that has been absent in previousfermentation systems. The container frame is used to provide processing,e.g., upstream or downstream processing in the same sample vessels.

[0047] The present invention provides a novel fermentor apparatus thatallows batch mode fermentation using a plurality of small samples. Forexample, small sample sizes overcome the disadvantage of sub-optimalgrowth rates and yields that exist in large batch mode processes. Inaddition, the present system eliminates the need for sample handling forpost-fermentation processing. The sample vessels in the presentinvention are used directly in any post-processing steps, whicheliminates many cleaning and sterilizing steps as well, therebyproviding a less expensive, more efficient, and faster fermentationprocess.

[0048] The present invention also overcomes the disadvantages of thecontinuous feed systems, e.g., with the small sample sizes used. Forexample, the estimated growth curves used in large scale continuous feedprocesses are unnecessary in the present invention. Therefore, theunpredictable results and frequent monitoring of continuous feedprocesses are not a problem in the present invention.

[0049] The present invention provides a fermentation apparatus thatsolves the above problems, e.g., by using small sample sizes andfermenting the multiple samples simultaneously. By simultaneouslyperforming multiple fermentations, e.g., small scale fermentations, inbatch mode, optimal mixing is achieved, optimal temperature and pH canbe maintained as well as many other advantages that will be apparentupon further reading of the present description.

I. A multi-sample Fermentation Apparatus

[0050] The present invention provides a multi-sample fermentationapparatus. Typically, the apparatuses of the invention comprise a sampleholder or container frame and a gas distribution system. For example, inone embodiment, a container frame is used to hold and/or transport anarray of sample vessels for fermentation. A fermentor head, e.g.,comprising an array of cannulas corresponding to the array of samplevessels, is coupled, e.g., directly, to the container frame and/orsample vessels. Gas is distributed into the multiple sample vessels viathe cannulas and fermentor head providing multi-sample fermentation.Various components of the apparatuses are described in more detail belowfollowed by methods of using the apparatuses and example fermentors.

[0051] A Container Frame is used to Hold a Plurality of Sample Vessels

[0052] A “container frame” as used herein refers to an arrangement thatholds and/or maintains a plurality of sample vessels in a desiredarrangement. Typically, the container frames of the invention aretransportable and autoclavable. In addition, they typically have nomovable parts. A transportable container frame is one that is easilytransported or moved while holding the sample vessels in the desiredarrangement. For example, a container frame of the invention optionallyhas handles for transportation to a processing station, e.g., afterfermentation is complete. An autoclavable container frame is one thatcan be placed directly in an autoclave for sterilization, e.g.,including the sample vessels and samples if desired.

[0053] By using a transportable container frame, the entire array ofsample vessels is optionally transported to and from one fermentationprocessing station to another processing station in a multiple processproduction. For example, a transportable container frame is optionallyused to transport an array of sample vessels into a temperaturecontrolled area such as a water bath, e.g., a water bath controlled by atemperature controller and temperature coil immersed in the water bath.Other forms of temperature control are also optionally used, such astemperature controlled gel baths, ovens, glove boxes, or air chambers.

[0054] Typically, the container frame maintains the sample vessels in anarray, e.g., a rectangular array. In an embodiment shown in FIG. 1,individual sample vessels 15 are configured in a rectangular array, butthe array is optionally configured in any physical construct that isconducive to fermentation or that is compatible with other processingsteps. For example, a honeycomb, circular, triangular, or linearconfiguration may be more efficient in other contemplated applicationsof the present invention.

[0055] The container frames of the present invention typically have aplurality of placement wells for positioning the plurality of samplevessels, e.g., in an array. For example, the placement wells optionallycomprise indentations in the bottom of a container frame, into whichsample tubes are optionally placed. In addition to the indentations orwells in the bottom of the container frame, the container framesoptionally include an upper portion, e.g., for supporting the tops ofthe sample tubes and maintaining their position. Example containerframes are shown in FIGS. 1 (container frame 250) and FIG. 13 (containerframe 1300).

[0056] For example, the bottom of each individual sample vessel istypically positioned within a placement well, e.g., placement well 257in FIG. 1 or placement well 1350 in FIG. 13. The array of placementwells preferably mirrors the configuration of the sample vessel arrayand is embedded in the transportable container frame. Placement wellsmay, however, be arranged in alternative configurations. For example,placement wells may be arranged as linear troughs, each holding a row ofsample vessels. In another embodiment, placement wells are absent fromthe transportable container frame. For example, the container frameoptionally has a solid bottom surface with no indentations or wells. Thesample vessels are then positioned in the frame, e.g., tightly packedagainst the sides of the frame to maintain the array configuration.

[0057] Sample vessels of the present invention typically comprise testtubes, other sample tubes, jars, flasks or any other container forholding a sample. Typically, the sample vessels have a volume of about50 to about 200 milliliters, more typically about 80 to about 100 ml.The sample vessels are typically placed in an array of placement wellsin a container frame, e.g., for autoclaving, processing, fermentation,and the like.

[0058] In some embodiments, the sample vessels are constructed of Pyrexglass or polycarbonate, but other suitable materials are optionally usedto construct the sample vessels. For example, plastic, ceramic, metal,e.g., aluminum, or any other material is optionally used that isnon-reactive to fermentation medium or to other materials involved inadditional processes contemplated in a multiple process production, suchas in a high throughput system. It will further be appreciated that thefermentation medium may be the same medium in each individual samplevessels or, alternatively, the array of sample vessels optionallyincludes a combination of different fermentation mediums. For example,fermentation medium may be the same in each individual sample vessel andcontain the same fermentation broth for a bulk synthetic process.Alternatively, each sample vessel in an array may have a slightlydifferent fermentation broth in order to optimize the production yieldof a certain component.

[0059] In some embodiments, sample vessels with gripper surfaces areoptionally used. In this embodiment, the container frame typicallycomprises a corresponding gripper surface, e.g., for maintaining thevessels in the desired configuration or to aid in transporting the arrayof sample vessels to and from a fermentation station and/or processingstation.

[0060] In other embodiments, sensors are optionally included in thesample vessels of the invention. For example, a pH or temperature sensoris optionally positioned proximal to or within a sample vessel tomonitor the fermentation reaction.

[0061] Fermentation samples are optionally placed in the sample vesselsprior to their placement in the container frame or after such placement.In one embodiment, colonization of bacteria and other preparative stepsare performed within the sample vessels, e.g., while they are containedin the container frame. For example, bacteria and initial nutrients aredispensed into each sample vessel at a prior processing station. Beingable to prepare bacteria directly in each individual sample vesseleliminates the need to inoculate a culture and initiate colonization ina separate container before transferring the sample to the fermentationapparatus. Using the container frame arrangement of the presentinvention to colonize the fermenting bacteria decreases costs byeliminating a separate colonization arrangement. Once bacteria arecolonized, sample vessels are conveniently transported, e.g., within thecontainer frame, to a fermentation station, e.g., a water bath or anyother temperature controlled area, such as a heated room. At thefermentation station or any time prior, a gas distribution arrangementis attached to the container frame to bubble gas into each sample vesselfor fermentation. The gas distribution arrangements are described inmore detail below.

[0062] A Gas Distribution Arrangement is used to Provide Gas to aPlurality of Sample Vessels

[0063] The gas distribution arrangement is used to provide gas flow tothe sample vessels during fermentation. The gas distribution systemtypically comprises a gas inlet which is configured to flow gas from agas source into a plurality of sample vessels in a container frame.Typically, the gas distribution arrangement is attached to the containerframe, e.g., placed on top or screwed down. For example, the gasdistribution arrangement typically comprises or is coupled to aplurality of cannulas through which the gas is flowed. The cannulasextend into each sample vessel for delivery of gas, e.g., to the bottomof the sample vessel. Such cannulas also optionally provide agitation ofthe sample within the sample vessel.

[0064] A gas source typically comprises a source of one or more gases,for example, air and oxygen. For example, in one embodiment the gassource contains an inlet for N₂ gas and an inlet for 02 gas. The ratioof each gas can be controlled either manually or remotely by using aprocess controller. The ability to adjust gas ratios enables the presentinvention to optimize the amount of gas, e.g., oxygen, needed as thegrowing conditions change throughout the fermentation. For example, aprocess controller is optionally used to linearly change the ratio ofair/oxygen over the course of a fermentation. Alternatively, the ratiois changed stepwise as fermentation proceeds. Any type, mixture, ornumber of gases are optionally introduced and mixed through the gassources of the invention and provided to fermentation samples containedin one or more sample vessels, e.g., through a set of cannulas.

[0065] A cannula is a small tube for insertion into a duct or vessel,e.g., a fermentation sample vessel or tube as provided herein. In thepresent application, the cannulas are positionable inside the pluralityof sample vessels, e.g., they typically comprise flexible or rigid tubesthat are inserted into sample vessels for the delivery of various gasesinto the sample vessels. In one embodiment, the cannulas are arrangedinto an array, which array typically corresponds to an array of samplevessels. An example array of the invention comprises an 8 by 12 memberarray of sample vessels each having an associated rigid cannula.Typically, a cannula extends substantially to the bottom of eachindividual sample vessel in order to increase aeration and mixing. Forexample, the cannula optionally extend about 15 to about 16 cm from thebottom surface of a gas distribution arrangement. In some embodiments,two or more cannulas are provided in each sample vessel.

[0066] In the embodiment illustrated in FIG. 8, gas flows throughcannula 22 through three passages. Gas flow through passages areoptionally individually or collectively regulated. Smaller gas bubblesare obtained with multiple small passages than with a single, largerpassage through the cannula. As a result, gas bubbles formed from thesemultiple passages have more surface area than bubbles formed from asingle passage. In a preferred embodiment, passages are precisiondrilled in order to more accurately adjust gas flow within each passageand to ensure uniform gas delivery across the set of sample vessels.Fewer or more passages may be used according to the specific applicationof the present invention. For example, the cannulas typically have about1 to about 5 passages, more typically, 2 or 3 passages. Passages areoptionally the same or different sizes and may be circular or anynon-circular shape, such as rectangular, oval, or triangular.

[0067] In one embodiment cannula are included in a cannula assemblycomprised of an array of individual cannulas corresponding to theplurality of sample vessels. Each individual cannula is optionallyconnected by a fastener which couples the cannula to a gas distributionarrangement.

[0068] Gas, e.g., oxygen or an oxygen/air mixture, is delivered, e.g.,from a manifold or other distribution system, to the sample vessels viathe cannulas, thus oxygenating, if desired, the entire array of samplevessels within the container frame. For example, a gas source isoptionally coupled directly to the gas distribution arrangement, e.g.,with or without the use of a manifold, as illustrated in FIGS. 6, 12,and 14.

[0069] In this manner, the exact mixture of gases delivered from the gassource is uniformly distributed to each individual cannula assembly. Anygas distribution arrangement is optionally employed that uniformlydelivers oxygen, an oxygen containing mixture, or another gas or gasmixture capable of fermenting the sample, from a gas source into theplurality of sample vessels. Example gas distribution arrangements areprovided in FIGS. 1, 3, 12, and 14, which are described in more detailin the examples provided below.

[0070] In some embodiments, the gas distribution arrangement iscomprised of one or more plates attached to an array of cannula, e.g.,using a manifold, and a gas inlet, which delivers oxygen, an oxygencontaining gas mixture, or another gas or gas mixture capable offermenting the sample, to the sample vessels via the cannula.

[0071] Typically, the plates are aligned and fastened together, e.g., toform an air-tight, liquid-tight seal. A hollow space or interior spacetypically exists between the plates or within one of the plates throughwhich gases are uniformly distributed to the associated cannula array.Any suitable fastener may be used. For example, guide pins, rivets,nails, nut/bolt combinations, or magnets may be used. A releasablefastener, such as a screw or nut/bolt combination, is used in apreferred embodiment, although permanent type fasteners, such asadhesives, may be desired in some applications. Vertical supports areoptionally attached to the gas distribution arrangement, thus supportingthe arrangement on an array of sample vessels.

[0072] The plates are optionally composed of any suitable material thatmaintains the structural integrity of the plate during fermentation. Forexample, a plate is optionally composed of metal, plastic, ceramic, orany suitable composite. In one example, the plates compriseTeflon™-coated aluminum, thus enabling the plates to undergo autoclavesterilization procedures along with the container frame and samplevessels as described above.

[0073] In one embodiment, the gas distribution arrangement comprises twoplates. The first plate, e.g., the bottom plate, typically comprises aplurality of sample vessel areas or indentations on the bottom surface.The indentations correspond to the array of sample vessels held in thecontainer frame and serve to cap the sample vessels. FIGS. 9-11illustrate features encompassed by the indentations, e.g., sample vesselarea or indentation 625 on bottom portion 646. The indentations orrecesses are also used, e.g., to immobilize the sample vessel within thecontainer frame. Although the indentations are illustrated as circular,they are optionally any shape, e.g., to correspond to a variety ofsample vessels.

[0074] One or more vents are typically positioned on the circumferenceof the sample vessel area, cap, or recess to allow gases and built uppressure to escape the sample vessel. FIG. 11 illustrates one embodimentof a venting space. However, other configurations of venting spaces andrecesses are optionally constructed such that built-up pressure withinsample vessels can escape without contaminating other sample vessels.

[0075] When the top surface of a sample vessel abuts the bottom surfaceof the gas distribution arrangement, gases, liquids, emulsions, orexcess pressure built up in the sample vessel escape through a recessand/or venting space created in the gas distribution arrangement.Cross-contamination of these escaping elements is significantly reducedbecause a vertical edge in the bottom surface of the gas distributionarrangement separates each sample vessel from an adjacent sample vessel.Moreover, gas flow from the cannulas maintains a positive pressurewithin the sample vessel such that contaminants outside a particularsample vessel are not drawn in through the vent.

[0076] In some embodiments, the first plate comprises the plurality ofcannulas that deliver gas to the sample vessels. The cannulas typicallyextend from the top surface of the plate, through the plate, and belowthe bottom surface of the plate. The cannulas are generally ofsufficient length to reach within about 1 cm to about 0.1 cm of thebottom of the sample vessels. The cannulas open to the top surface ofthe plate, e.g., for gas to be distributed through the cannulas into thesample vessels. The cannulas are configured to be positionable in anarray of sample vessels, e.g., held in a container frame.

[0077] In addition to the cannulas, the first plate optionally includesa plurality of apertures that correspond to the array of sample vessels.For example, the apertures optionally provide an opening through thefirst plate, through which fluids may be added into the sample vesselswhen the gas distribution arrangement is attached to a container frame.

[0078] The first plate is typically attached to a second plate, e.g.,with screws or adhesives, which second plate typically comprises one ormore gas inlets for providing gas flow into the cannulas of the firstplate. The gas inlet opens into an interior space created between thesecond plate and the first plate, which interior space provides gas flowto the cannulas.

[0079] In addition, the second plate also comprises a plurality ofapertures, e.g., to provide liquid access to the sample vessels. Theapertures of the second plate typically align with or match theapertures on the first plate when the two plates are coupled. Theapertures provide openings through which liquid can be added into thesample vessels in a container frame attached to the gas distributionarrangement. The apertures also serve as openings for an array ofaspirators or dispensers that can be used to aspirate or dispense liquidinto the sample vessels. In other embodiments, pipettes or syringes areused to draw samples or add nutrients, water, etc, e.g., through theapertures. The gas distribution arrangement also optionally comprises alid for covering the apertures when a sealed environment is desired. Thefirst plate and second plate together comprise a fermentor head ormanifold for delivering gas or fluid to a plurality of sample vessels.More detailed examples are provided below.

[0080] A process controller is also optionally coupled to thefermentation apparatus of the invention, e.g., for controlling gas flowto the cannulas, for altering ratios of air to oxygen that are bubbledthrough the cannulas, for monitoring and controlling temperature, fordirecting the addition of various reagents, and the like. An automatedprocess using a process controller is described in more detail in theexamples below.

[0081] Other devices are also optionally coupled to the fermentorapparatus of the present invention. For example, dispensers, aspirators,centrifuges, and other processing devices are optionally coupled to thefermentor or configured for use with a container frame, e.g., so thatsamples can be processed in the same vessel in which fermentation iscarried out. For example, a dispenser is optionally configured todispense liquid into a plurality of sample vessels held in a containerframe, e.g., through a plurality of apertures in a gas distributionarrangement. Aspirators are likewise optionally configured to coordinatewith the container frame and gas distribution manifolds of the presentinvention.

[0082] A centrifuge is also optionally used in processing fermentationsamples. For example, a centrifuge is optionally configured to acceptthe sample vessels as centrifuge tubes to avoid transferring of samplesprior to centrifugation. For more information on centrifugation systemsfor use in the present invention, see, e.g., U.S. Ser. No. 09/780,589,entitled “Automated Centrifuge and Method of Using Same,” by Downs etal, filed Feb. 8, 2001.

II. Methods of Fermenting Samples in a Multi-sample Fermentor

[0083] The multi-sample fermentors described above are used forsimultaneously fermenting a plurality of samples, e.g., in a containerframe that is transportable, e.g., to a processing station. The presentinvention also provides methods of using such fermentors, e.g., inconjunction with one or more processing steps. For example, the methodsprovided typically comprise providing a plurality of sample vessels in acontainer frame, each of which sample vessels contains a sample of about50 to about 100 milliliters, more typically 65 ml. The samples arefermented in the sample vessels within the container frame.

[0084] Fermentation is used herein to refer generally to any process inwhich cells are used to convert raw materials, e.g., water, air, sugars,mineral salts, nitrogen sources, and the like, or enzyme substrates intodesired products, e.g., proteins. Types of cells used include, but arenot limited to, animal cells, yeast cells, and bacterial cells, e.g., E.coli, Bacillus, and the like. The cells are typically grown in a growthmedium and then products are harvested. Fermenting typically involvessimultaneously delivering gas to each of the sample vessels through aplurality of cannulas associated with the sample vessels, e.g., to aidgrowth of the cells. For example, the methods typically compriseattaching a fermentor head as described herein to a container framecontaining the plurality of samples to be fermented. Once fermented, thesamples are transferred to a post-processing station, e.g., acentrifuge. Typically, the post-processing station is configured toaccept the same sample vessels in which the samples were fermented. Inaddition, some processing stations are configured to receive thecontainer frame containing the sample vessels, e.g., a dispensing oraspirating station. An example method is described below and in FIGS. 4and 5.

[0085]FIG. 4 describes fermentation method 300 practiced in accordancewith the present invention. Block 310 provides for a plurality of samplevessels 15. By providing a number of smaller volume fermentationvessels, this method is more advantageous than production scalefermentation methods that use bulk fermentation vessels, in that smallervolumes of growth medium are more predictable in their yield andnutrient needs than are standard production scale volumes that areutilized in bulk fermentation methods. The number of sample vessels thatmay be fermented at any one time is unlimited by the present invention,and instead is only limited either by the configurational practicalitiesof any one fermentation apparatus or by the number of sample vesselsthat may be handled by further processing steps in the production.

[0086] Block 315 arranges a plurality of sample vessels into an array,e.g., a rectangular 8 by 12 array. However, the array is optionallyconfigured in any shape that is practicable for the fermentationapparatus. For example, sample vessels are optionally arranged in arectangular array, a honeycomb configuration, or a linear array.

[0087] Block 320 arranges a plurality of cannula into an arraycorresponding to the sample vessels. According to the present invention,each cannula in this cannula array corresponds to an individual samplevessel in the sample vessel array, which are arranged in block 315. Inone embodiment, the plurality of cannula is limited by the number ofsample vessels arranged in block 315.

[0088] Block 325 creates a gas distribution arrangement for deliveringoxygen and/or one or more other gases to a fermentation media in thesample vessels. For example, one embodiment fastens a cannula array to agas distributor, which is connected to a manifold. The cannula array maybe fastened by any means achieving a liquid-tight seal. For example,cannula are optionally connected via a union connector to a gasdistributor. Alternatively, cannula are pneumatically connected to thedistributor, or the cannula array and gas distributor are optionallymolded as a single unit. In another embodiment, the distributor connectsdirectly to a gas source without using a manifold. The methods ofcreating a gas distribution arrangement are optionally achieved usingany method of uniformly delivering oxygen and/or one or more other gasesfrom a gas source to a gas distributor such that gas is delivered toeach individual sample vessel selectively or collectively by way of acorresponding cannula.

[0089] Block 330 transports the container frame containing the pluralityof sample vessels to a temperature controlled area. Other methods knownto those of skill in the art for controlling temperature are alsocontemplated within the present invention. For example, the containerframe is optionally transported to a heated gel bath or a controlledtemperature room used to maintain a constant temperature.

[0090] Block 335 positions the gas distribution arrangement created inblock 330 on top of the container frame, e.g., using screws or by merelybeing placed on top and held in position by a groove assembly as shownin FIG. 14. From this configuration, the array of sample vessels isfermented in block 340.

[0091] Once fermentation is complete, block 345 removes the gasdistribution arrangement from the container frame. The sample vesselsare optionally transferred from the container frame directly to apost-fermentation processing station in block 350, e.g., by manipulatinga gripping surface located on each sample vessel. This post-fermentationprocessing station includes any processing step where the fermentationproduct may be processed directly from the sample vessel. For example,the array of sample vessels may be transferred, either manually orrobotically, from the container frame directly to an automatedcentrifuge. Alternatively, sample vessels may be transferred to anaspirating station or detecting station. In other embodiments, thesample vessels are not removed from the container frame but remain in itfor further processing, such as dispensing or aspirating, using adispenser or aspirator configured to coordinate with the array of samplevessels in the container frame.

[0092] In block 350, the fermentation product in the sample vessels isdirectly transferred into a post-fermentation processing station and inblock 355 the fermentation product is directly processed in the samplevessels themselves. For example, in one embodiment, sample vessels aretransferred directly to a centrifuge station in which the sample vesselsare positioned directly inside the centrifuge such that the samplevessels act as centrifugation tubes and the fermentation product iscentrifuged according to methods known in the art. Further processingsteps such as aspirating, reagent dispensing, or detecting alsooptionally occur directly in the sample vessel used in the fermentationprocess. In this way, the fermentation vessel provides a sample vesselthat holds the sample throughout the entire production process, therebyeliminating excess waste from transferring sample material from samplevessel to sample vessel as well as decreasing the cost of washing andsterilizing a fermentation apparatus in addition to sample vessels fromeach production process step. Other multiple process productions oranalyses may also be practiced in accordance with the present invention.

[0093] In FIG. 5, block diagram 400 shows how the present invention isintegrated into a multiple step, multiple process production. Block 410depicts a processing station prior to fermentation. In one embodiment,fermentation broth and fermentation nutrients are added to samplevessels at prior processing station 410. Other processing steps involvedin a multiple step production or analysis are also contemplated inaccordance with the present invention. For example, bacteriacolonization may occur in sample vessels at prior processing station410. Example preprocessing steps include, but are not limited to,deionization, e.g., of solvents, pasteurization of materials, andmixing, e.g., of cell nutrient broths and the like. Such steps aretypically used to process the raw materials, such as water, cell broths,sugars, nitrogen sources, and the like, used for the fermentation.Transporter 420, e.g., a robot, a technician, a conveyor belt, or thelike, is optionally used to transfer the sample vessels from processingstation 410 to a fermentation apparatus such as fermentation apparatus100. Other embodiments of a fermentation apparatus practiced inaccordance with this invention may also be used. For example, thefermentation apparatus shown in FIG. 14 or in FIG. 1 is optionally used.

[0094] It will further be appreciated that transporter 420 may transferthe sample vessels individually, in groups, or in an array configuredfor the fermentation apparatus. For example, in one embodiment, acontainer frame transports the sample vessel array to fermentationapparatus 100. Similarly, after fermentation, transporter 430 transportssample vessels from a fermentation apparatus to a post-fermentationprocessing station 410. In one embodiment, transporter 430 transports acontainer frame holding an array of sample vessels to a centrifugeprocessing station 410. Post-processing station 410 is optionally anyother processing step occurring in a multiple process or analysis, suchas an aspirating step, a dispensing step, or a detecting step. Examplepost-processing steps include, but are not limited to, precipitation,deionization, chromatography, evaporation, filtration, centrifugation,crystallization, drying, and the like. These steps are generallydirected to purification, retrieval, and concentration of materialsproduced in the fermentation. In this manner, multiple processing stepsare executed on each sample contained in the same sample vessel, thusenabling fermentation processes to be incorporated into high throughputor other multiple process systems. Example fermentation conditions aredescribed below.

[0095] The present invention preferably uses fermentation conditionsthat lead to high level production of soluble proteins. Thesefermentation conditions may employ the use of high levels of yeastextract and bactotryptone (rich media, referred to as terrific broth orTB). Secondly, this media is optionally supplemented with 1% glycerol(additional carbon source). Lastly, the media preferably is typicallybuffered with 50 mM MOPS. Alternatively, a defined media comprisingamino acids and 50 mM phosphate as opposed to MOPS is used. The firsttwo additions allow the cells to be grown for up to about 10 hourswithout apparent loss of nutrients. The highly buffered media preventsthe cells from being exposed to high levels of acid (low pH) whichroutinely occurs during fermentation.

[0096] Surprisingly less than 5% of human proteins expressed in normalLuria Broth or LB media, are typically found to be soluble. However,using the above media, 15-20% of human proteins expressed in E. coli nowappear to be soluble.

[0097] In a preferred embodiment, the fermentation media is prepared asfollows. TB media is prepared in 7 L batches. Antibiotics are not addedto TB media until the day it will be used for a fermentation run. Toprepare the 7 L bath, the following steps are performed: (1) Fill aclean 10 L pyrex bottle with ˜4 L DI H₂O or 18 megohm water, add a largestirbar; (2) Add 168 g Yeast Extract; (3) Add 84 g Tryptone; (4) Add 70ml Glycerol; (5) Stir on stirplate until completely dissolved; (6) QS to6.3L, e.g., with 18 megohm water; (7) Autoclave on the longest liquidcycle. Remove TB media from the autoclave as soon as possible, e.g., toprevent carmelization or burning of the carbon source and/or to allowfor a quick cool down; (8) Store TB media at room temperature; and (9)Record process. TB Media is the same for all fermentor runs. However,Fermentor Media is not necessarily the same for all runs. For example,one difference in media is the antibiotic(s) added just beforefermentation. On the same day of a fermentation run, the following maybe added to TB media: (1) 350 mls of 1 M MOPS pH 7.6; (2) 7 ml Antifoam;(3) 7 ml 20 mg/ml Chloramphenicol; (4) 7 ml 100 mg/ml Ampicillin; (5)Add enough 18 megohm H₂O to bring the volume up to 7 L; (6) Writeeverything added to TB media on its label; (7) Cap tightly and shakebottle well; and (8) Record process. The above medium is only one ofmany possible choices known to those of skill in the art, which areoptionally used with the present fermentors and methods.

[0098] When fermentation is complete, the sample vessels are transferredto a post-processing unit as described above, e.g., in the containerframe, either manually or robotically. For example, a robot optionallyremoves the sample vessels from the container frame and places them,e.g., in a centrifuge.

III Examples Fermentation Systems

[0099] Example Fermentor #1

[0100] In accordance with the present invention, an example fermentationapparatus is provided in FIG. 1. Fermentation apparatus 10 generallycomprises sample holder arrangement 255, cannula assembly 80 and gasdistribution arrangement 270. The illustrated fermentation apparatus 10is configured to separately and simultaneously ferment multiple batchsamples in sample vessels that are compatible with direct pre- andpost-fermentation processing as described above.

[0101] Sample holder arrangement 255 is comprised of gripping surfaces17, individual sample vessels 15, which typically form an array ofsample vessels, such as array 110, a transportable container frame 250,and an array of placement wells 260 corresponding to array 110. Grippingsurfaces 17 are optionally located on each individual sample vessel 15,which collectively form sample vessel array 110. It is preferable thatgripping surface 17 resides on the bottom of each sample vessel, butgripping surface 17 is optionally located on any surface of the samplevessel that enables sample vessel 15 to be transferred to or fromcontainer frame 250 or another processing station.

[0102] The bottom of each individual sample well 15 is positioned withina placement well,.e.g., placement well 257. The array of placement wells260 preferably mirrors the configuration of array 110 and is embedded intransportable container frame 250.

[0103] By using transportable container frame 250, the entire array ofsample vessels 110 is optionally transported to and from onefermentation processing station to another processing station in amultiple process production. In this illustrated example, transportablecontainer frame 250 transports array of sample vessels 110 into atemperature controlled area 210 such as a water bath. In thisembodiment, temperature controlled area 210 is comprised of water bath240 in water bath container 215, which is controlled by water bathtemperature controller 220 and temperature coil 230 immersed in waterbath 240.

[0104] In FIGS. 1-3, an example gas distribution arrangement is shown.Gas distribution arrangement 270 is comprised of gas source 85 connectedto manifold 75. Conduit 70 connects manifold 75 to connector 65.Connector 65 connects manifold 75 to gas distributor 55.

[0105] In the embodiment illustrated in FIGS. 1 and 3, cannula assembly80 is comprised of cannula array 120, which is composed of individualcannulas 22 that correspond to sample vessel array 110. Each individualcannula 22 is optionally connected by a fastener 35, which couplescannula 22 to a gas distribution arrangement 270. Cannula 22 preferablyextends substantially to the bottom of each individual sample vessel 15in order to increase aeration and mixing.

[0106] In another embodiment, each individual cannula is attacheddirectly to gas distribution arrangement 270 in an airtight,liquid-tight manner. Eliminating the need for a fastener, thisembodiment directly integrates cannula 22 into gas distributionarrangement 270, thereby decreasing the number of surfaces, grooves,and/or pockets available for possible bacterial contamination, and thusdecreasing the opportunities for fermentation spoilage. Likewise,cannula 22, when integrated into a gas distribution arrangement 270 areoptionally autoclaved with gas distribution arrangement 270, therebyeliminating the need to unfasten each cannula 22 separately beforecleaning and sterilization. This convenience saves both time and moneyas well as adding to the uniformity of each batch. For example, thepossibility for human error is minimized, because each cannula 22 doesnot have to be fastened individually before each fermentation run orunfastened individually prior to cleaning and sterilization. Also anynon-uniformities in any one cannula 22 will be immediately apparent asan individual cannula 22 will be constantly associated with the samesample vessel in each run. Integrated cannula are shown in FIG. 14.

[0107] Referring to FIG. 3, gas, e.g., oxygen, is delivered frommanifold 75 to all parts of distributor 55 through a hollow space 60 ofdistributor 55, thus oxygenating, if desired, the entire array of samplevessels 110. Oxygen and/or one or more other gases is delivered fromdistributor 55 through individual cannula 22, which is connected todistributor 55 by way of cannula assembly 80.

[0108] In one embodiment, cannula assembly 80 is comprised of aconnector 45 on an inside face of distributor 55 as well as connector 40on an outside face of distributor 55. Fastener 35 attaches individualcannula 22 to connector 40 on distributor 55. Arrows 25 depict oxygenand/or one or more other gases flowing from cannula 22 into fermentationmedium 20 and producing gas bubbles 30. For example, gas source 85 isoptionally coupled directly to dispensing plate 645 without the use ofmanifold 75, as illustrated in FIGS. 6 and 12. Likewise, cannulaassembly 80 may be constructed by alternative methods. For example, asshown in FIG. 12, cannula 22 is attached directly to dispensing plate645.

[0109] In this manner, the exact mixture of gases delivered from gassource 85 is uniformly distributed to each individual cannula assembly80. Any gas distribution arrangement is optionally employed thatuniformly delivers oxygen, an oxygen containing mixture, or another gasor gas mixture capable of fermenting the sample, from gas source 85 intosample vessel 15.

[0110]FIGS. 6 and 12 illustrate another embodiment of a gas distributionarrangement. Gas distribution arrangement 270 is comprised of adispensing plate 645 directly attached to an array of cannula 120, thatis configured without a manifold, manifold conduit, or manifoldconnector. In this embodiment, dispensing plate 645 is comprised of abottom portion 646 and a top portion 647 (not shown). Inlet 630 deliversoxygen, an oxygen containing gas mixture, or another gas or gas mixturecapable of fermenting the sample, to dispensing plate 645 from gassources 85 (not shown).

[0111] Bottom portion 646 and top portion 647 are aligned and fastenedtogether through apertures 640, e.g., to form an air-tight, liquid-tightseal. A hollow space exists between portions 646 and 645 through whichgases are uniformly distributed to cannula array 120. Apertures 635 areused to fasten vertical supports to dispensing plate 645 that allowdispensing plate 645 to rest adjacent to array of sample vessels 110.Any suitable fastener may be used. In the illustrated example, screwsconnect upper portion 647 and bottom portion 646 to form dispensingplate 645. Screws also fasten aluminum legs to dispensing plate 645 asvertical supports.

[0112] FIGS. 9-11 illustrate yet another embodiment of a gasdistribution arrangement. In this embodiment, cannula 22 is directlyattached to bottom portion 646. Aperture 620 holds a dispensing tube 760(not shown) for dispensing nutrients and other solutions into samplevessel 15. Aperture 620 is optionally used to access samples during thefermentation process, using, e.g., pipettes or syringes to draw samplesor add nutrients, water, and/or the like into the sample vessels.Fastening groove 650 enables dispensing tube 760 to be fastened todispensing plate 645. Indentation 655 and vertical edge 665 create acircular recess that helps immobilize sample vessel 15 within samplevessel area 625. Although in this embodiment, indentation 655 iscircular and corresponds to the shape of sample vessel 15, othersuitable shapes may be used.

[0113] Vent 610 is positioned on the circumference of sample vessel area625 and allows gases and built up pressure to escape sample vessel 15.Referring to FIG. 11, vent 610 creates venting space 675. Becausevertical edge 670 is larger than vertical edge 665, venting space 675occupies a deeper recess than recess 655. The difference in heightbetween vertical edges 670 and 665 is equal to the height of verticaledge 680 and determines the depth of venting space 675. Otherconfigurations of venting space 675 and recess 655 (and, accordingly,vertical edges 665, 670, and 680) may be constructed such that built-uppressure within sample vessel 15 can escape through venting space 675without contaminating other sample vessels.

[0114] When the top surface of sample vessel 15 abuts surface 660,gases, liquids, emulsions, or excess pressure built up in sample vessel15 may escape through recess 655 and venting space 675.Cross-contamination of these escaping elements is significantly reducedbecause vertical edge 670 separates sample vessel 15 from an adjacentsample vessel 15. Moreover, gas flow from cannula 22 maintains apositive pressure within sample vessel 15 such that contaminants outsidesample vessel 15 are not drawn in through venting space 675 into samplevessel 15 by way of recess 625, 655, or 675. Other vents 610 may beconfigured such that excess gases, liquids, emulsions, or excesspressure may escape through vent 610 without cross-contaminating othersample vessels 15.

[0115] In another embodiment of gas distribution arrangement 270,illustrated in FIG. 2, array 110 is configured such that gasdistribution arrangement 270 oxygenates, for example, each individualsample vessel 15 as opposed to utilizing a dispensing plate 645. Thus,array of sample vessels 110 is optionally oxygenated (or provided withother appropriate gas) collectively or individually by adjusting cannulaassembly 80 for any individual sample vessel 15. For example, in oneapplication, section A may be oxygenated (or provided with otherappropriate gas) twice as long as section B.

[0116] In the illustrated example, cannula array 120 corresponds tosample vessel array 110, which is composed of individual sample vessels15. Each individual sample vessel 15 also corresponds to an individualcannula assembly 80 which is connected to distributor 55. Oxygen and/orone or more other gases are delivered to distributor 55 through manifoldconnector 65. Oxygen and/or one or more other gases may be deliveredthrough each cannula assembly 80, or selectively to certain assemblies80. For example, cannula assemblies 80 in sections A and B may beutilized, while no gases flow to sections C and D.

[0117] Referring to FIGS. 3 and 12, gripping surface 17 allows forautomated or manual transfer of sample vessel 15 to and from thefermentation apparatus or another processing station, e.g., uponconclusion of fermentation. In one embodiment, gripping surface 17 ismagnetic such that a magnet attracts gripping surface 17 and transfersthe sample vessel to another processing station. In another embodiment,a gripping mechanism grips the outer sides of the sample vessel toeffect transfer. In yet another embodiment, gripping surface 17 is a lipat the top of the sample vessel. Other surfaces that may be gripped inorder to transport the sample vessel to or from the fermentationprocessing station are within the scope of the present invention. Forexample, gripping surface 17 is optionally on the inside, outside, topor bottom of sample vessel 15. In other embodiments, the samples areheld in place and transported with the aid of a gripper structure.

[0118]FIG. 12 illustrates one embodiment of a gas distributionarrangement. Gas distribution arrangement 270 and cannula 22 are usedtogether to provide gas to a sample vessel. In this example, oxygen, amixture of oxygen and other gases, or another gas or gas mixture isintroduced into dispensing plate 645 through inlet 630. Fasteners suchas screws connect and align upper portion 647 to bottom portion 646through apertures 640. Dispensing tube 760 and cannula 22 are directlyattached to dispensing plate 645 and can be replaced by unfasteningportions 646 and 647, replacing either or both dispensing tube 760 orcannula 22, and refastening portions 646 and 647. It is preferable fordispensing tube 760, cannula 22, inlet 630, and portions 646 and 647 toremain fastened together such that these elements are autoclaved as oneunit. This allows for significant sterilization without the time andcost expense of dismantling arrangement 270 after each fermentation inorder to separately sterilize each element.

[0119] In the illustrated example, a top surface of individual samplevessel 15 abuts directly onto surface 660 within sample vessel area 625.The top surface of sample vessel 15 is positioned within recess 655.Surface 660 preferably is not in contact with the entire circumferenceof the top surface of sample vessel 15. Also preferably, vent 610 ispositioned adjacent to surface 660 such that a gap 672 exists betweensurface 660 and the vertical edge of sample vessel 15, thereby creatinga passage for excess gases, emulsions, or pressure to escape from samplevessel 15 through venting space 675. Gas flow through cannula 22provides sufficient pressure such that contaminants are not drawn intosample vessel 15 through venting space 675.

[0120] Example Fermentor #2

[0121] FIGS. 13-21 illustrate another embodiment of the fermentorapparatus of the present invention. Generally, the apparatus comprises acontainer frame comprising placement wells, and a gas distributionarrangement comprising a cannula array. Each piece is described in moredetail below and by reference to the figures.

[0122] Container frame 1300, as shown in FIG. 13, comprises bottom 1310and top portion 1320 connected by side portions 1325 and 1330. Thecontainer is easily transportable, e.g., by grasping handles 1335 and1340 which are attached to sides 1325 and 1330. Each side 1325 and 1330has two grooves 1345 which can each receive a pin for securing a gasdistribution arrangement, such as that shown in FIG. 16, e.g., usingpins 1480. Top portion 1320 and bottom portion 1310 together form anarray of placement wells 1350. Bottom portion 1310 of the containerframe has a plurality of indentations that serve as bottoms for theplacement wells, in which sample vessels are placed. For example,container frame 1300 comprises an 8 by 12 array of placement wells. Topportion 1320 comprises a matching array of holes 1360 which holesreceive the sample vessels into the container frame and hold them inposition within the container frame. Together holes 1360 andindentations 1355 in container frame 1300 form a rack for holding aplurality of sample vessels, e.g., tubes. Although holes 1360 are shownas circles, the shape is optionally configured to receive any desiredsample vessel.

[0123]FIG. 14 illustrates a gas distribution arrangement coupled tocontainer frame 1300. The gas distribution arrangement comprises fourpins 1480 which slide into grooves 1345 to hold the gas distributionarrangement in place over the container frame. As shown in FIG. 14, thegas distribution arrangement comprises first plate 1465 and second plate1470, which are typically fastened together, e.g., using screws or pins.An optional lid, e.g., lid 1460, is also shown. In addition, the gasdistribution arrangement comprises handles 1410 and 1420 attached tosecond plate 1470 for easy positioning and removal of the gasdistribution arrangement.

[0124] Inlets 1430 and 1440 provide gas inlets to the gas distributionarrangement, which gas inlets typically receive gas from a gas sourceand deliver it, e.g., to a plurality of cannulas. Typically, theplurality of cannulas is attached to the gas distribution arrangement,e.g., as part of the first plate. For example, in the illustratedembodiment, cannula 1450 is part of first plate 1465 and extends fromthe top of the first plate, through the first plate and below, such thatthe cannula is positionable inside a placement well, e.g., well 1350, orinside a sample vessel positioned within placement well 1350.

[0125] Typically, first plate 1465 comprises the cannula array and aplurality of apertures. The apertures of the first plate align with aset of apertures on the second plate to provide access to the samplevessels within the placement wells. The cannula array is optionallymolded as part of the first plate or separately formed and then attachedto the first plate. For example, an additional set of apertures isoptionally present in the first plate to accept the array of cannula,e.g., which are received into the aperture and secured using o-rings.

[0126]FIG. 18 illustrates the bottom surface of first plate 1465. Forexample, on the bottom surface of the first plate, an array of samplevessel areas 1810 or indentations are used to cap the sample vessels andprovide venting space as described above in Example 1. Each samplevessel area comprises an aperture to provide access to the sample vesselpositioned with the associated placement well, a cannula associated witheach placement well for delivering gas into each sample vesselpositioned within the well, and a vent for relieving pressure build upduring fermentation. In addition, FIG. 18 illustrates apertures 1830 and1840, which are used, e.g., to attach the second plate to the firstplate, e.g., via a set of screws. FIG. 19 provides a detail drawing of aportion of FIG. 18 illustrating aperture 1920, vent 1930, and cannula1940. In addition, FIG. 19 illustrates gasket or o-ring 1950 that servesto provide a seal between the first and second plates.

[0127] Second plate 1470 typically comprises a set of apertures asdescribed above, which correspond to the set of apertures in plate 1465.These apertures are used, e.g., for liquid dispensing and/or venting.The apertures in the two plates connect to form a passageway thatextends through both plates for access to placement wells 1350. Theapertures are closed off from the interior space and can be capped usinga lid as shown in FIG. 14 when a sealed system is desired. In addition,second plate 1470 typically comprises the gas inlet, e.g., inlet 1430,and an interior space through which gas is flowed. FIG. 21 provides aside view of the gas distribution arrangement as shown in FIG. 14. Forexample, FIG. 21 shows cannulas 1450 extending below the first plateinto the placement wells and apertures 1920 extending through the firstplate and the second plate.

[0128]FIG. 20 illustrates a cross-sectional view of the gas distributionarrangement of FIG. 14, which comprises a first and a second plate. Topplate 1470 is attached to bottom plate 1465, e.g., using screwspositioned through apertures 1830, and 1840. The first plate, which ison the bottom, comprises apertures 2010 and cannulas 2020. The aperturesare open holes in first plate 1465, which align with similar aperturesin second pate 1470, the top plate. The cannula are inserted into thefirst plate through another set of apertures secured with 0-rings, e.g.,to form a seal between the top and bottom plates. The cannulas extendfrom the top surface of plate 1475 into placement wells 1350 such thatthey are easily positioned in an array of sample vessels held in theplacement wells. Cannula 2020 does not extend into plate 1470, but abutsit. Adjacent to where cannula 2020 abuts plate 1470 is venting space2030 which couples the cannula to interior space 2040 of the top platethrough which interior space gas flows in through an inlet, e.g., inlet1430.

[0129]FIG. 15 illustrates a container frame with a liquid additionmanifold assembly coupled to it. Container frame 1300 is shown withfirst plate 1465 positioned on top using pins 1480. Second plate 1470 ispositioned on top of the first plate and liquid addition manifold 1510is shown on top of the second plate of the gas distribution system. Theliquid addition manifold is optionally used to add liquid into thesample vessels, e.g., through corresponding sets of apertures in thefirst and second plate. FIG. 16 illustrates liquid addition manifold1510 in more detail, e.g., apertures 1620, which align with apertures onthe first and second plates of the gas distribution arrangement.Apertures 1620 are used to deliver liquid reagents into the samplevessels contained in the apparatus. Manifold 1510 is placed, e.g., usingpins, on top of the gas distribution system. In addition, FIG. 17, across-sectional view of the liquid addition manifold along line A-A,illustrates how pipettes or additional cannulas are used to dispenseliquid into the sample vessels.

[0130] Example System 3—an Automated System

[0131]FIG. 7 illustrates an example of an automated fermentationapparatus. Process controller 705 monitors and controls variouscomponents of apparatus 700 and preferably is a programmable computerwith an operator interface. Alternatively, process controller 705 is anysuitable processor that coordinates multiple components of apparatus700, such as timing mechanisms, adding solutions, adjusting temperature,adjusting gas flow rates and gas mixtures, detecting measurements,and/or sending an alarm or notification prompting operator intervention.Electronic couples 710, 755, and 795 connect various components offermentation apparatus 700 to process controller 705. For exampleelectronic couple 710 enables controller 705 to start, stop, and monitorsolution flow from feed solutions 720, 735, and 745. Likewise,electronic couple 775 enables controller 705 to start, stop and monitorreagent dispensing into sample vessels 15. Electronic couple 795 alsoenables controller 705 to transmit and receive information from sensors790 as well as monitor and adjust temperature controlled areas. Othercoupling devices are also optionally used in the present invention.

[0132] In one embodiment of fermentation apparatus 700, feed solutions720, 735, and 745 are pumped (either singly, in combination,sequentially, or collectively) from individual feed tubes 725 intodispensing tube 715. Selecting the appropriate solenoid determines whichfeed solution is pumped through dispensing tube 715. For example,solenoid 730 controls flow from feed solution 720 through feed tube 725.In another application, a mixture of feed solutions 720 and 735 aresimultaneously pumped into dispensing tube 715. In another application,feed solution 720 is fed into dispensing tube first, followed by anincubation period (directed by controller 705), followed by feedsolution 735 being pumped into dispensing tube 715. Differentcombinations of feed solutions are optionally used and more or fewerfeed solutions may be used with apparatus 700 according to any desiredapplication.

[0133] Using pump 710, which is optionally a peristaltic pump,dispensing tube 715 transfers feed solution to an individual dispensingtube 760. Each individual dispensing tube 760 corresponds to anindividual sample vessel 15 and tube 760 is positioned such that feedsolution 720, for example, is transferred volumetrically from dispensingtube 760 into its corresponding sample vessel 15 once solenoid 765 isopened. Each solenoid 765 corresponds to an individual sample vessel 15.Volumetric dispensing of feed solutions is controlled by processcontroller 705 which preferably controls the amount, the rate and thetime of dispensing. Dispensing tube 760 is optionally composed ofplastic, metal, or any material that is non-reactive to the feedsolution being dispensed.

[0134] In one embodiment; delivery solenoids 765 work in conjunctionwith pump 710 and controller 705 to deliver multiple feed solutions suchas feed solutions 720, 735, and 745 into individual sample vessels 15.Each solenoid 765 corresponds to a sample vessel 15 and the solenoids765 are manifolded together and fed by the output of a singleperistaltic pump 710. Each solenoid 765 preferably opens sequentially inorder to dispense a volumetric amount of feed solution 720. However,parallel addition is also contemplated within the present invention.

[0135] In one embodiment, feed solution 720 introduces nutrients intofermentation medium 20 through dispensing tube 715 using pump 710 andsolenoid 765 to deliver solution 720 to individual dispensing tube 760.After addition of feed solution 720, solenoid 730 is closed and solenoid740 corresponding to rinse solution 745 opens. Pump 710 delivers rinsesolution 745 through dispensing tube 715, thereby rinsing dispensingtube 715 with solution 745, which is then flushed into waste container785. Solenoid 780 controls flow from dispensing tube 715 into wastecontainer 785. Feed solution 735 is then pumped through dispensing tube715 and dispensed through tube 760. Dispensing tube 715 is rinsed againwith rinse solution 745 before another addition. Solenoids 765 arepreferably located very near to dispensing tube 760 in order to minimizedead volume downstream. In this way, dispensing tube 715 accuratelydelivers a known amount of feed solution 720 and 735 without crosscontaminating or fouling the next or different addition of feed solution.through dispensing tube 715. Accordingly, each addition isvolumetrically precise with a minimal, known amount of feed solutionfrom a previous addition diluting the next addition. In this way, feedsolutions such as additional nutrients, trace minerals, vitamins,sugars, carbohydrates, nitrogen containing compounds, evaporatingliquids, pH balancing compounds, buffers, and other liquids may be addedto fermentation media 20 in an automated, yet highly precise manner.

[0136] Coordinated by process controller 705, various components may beactivated either at pre-determined time intervals or in response to themeasurement of some physical property within sample vessel 15. Forexample, in one embodiment, an operator programs process controller 705to incubate sample vessels 15 for a pre-determined time period at aparticular temperature, add a desired amount of feed solution 720, andincubate further for another pre-determined time period at a differenttemperature. Any suitable combination of fermentation conditions may beprogrammed into process controller 705, which optionally comprises acomputer, computer network, other data input module, or the like.

[0137] In a preferred embodiment, process controller 705 coordinatestemperature control, the addition of feed solutions, adjustment of gasrates and gas mixtures, incubation periods, and rinsing in response todata received from sensors 790. Sensors 790 are optionally locatedinside or outside of individual sample vessels 15. Sensors 790 candetect color changes spectrophotometrically, monitor evaporation rates,measure changes in optical density, detect light changesphotometrically, detect pH changes, electrolytically measure redoxpotentials, monitor temperature fluctuations, or detect other physicalchanges and transmit this data to process controller 705. In response,process controller 705 accordingly adjusts various components ofapparatus 700. For example, by measuring the redox potential, sensors790 detect when a fermentation sample is being over-oxygenated orover-provided with another gas and process controller 705 accordinglyadjusts the gas flow or gas mixture ratio. As another example, processcontroller 705 can respond to a change in pH, as detected by sensors790, by adding a pH buffer from feed solution 720. In one embodiment,maximum protein expression may be detected by monitoring light emission,at which point fermentation is halted to minimize wasting fermentationresources after optimum fermentation yield has been reached.

[0138] Because of the uniformity of each fermentation medium 20, cannula22, and dispensing of feed solutions 720, very few, for example, one,sensor 790 is all that is necessary to monitor the entire array ofsample vessels 110. Alternatively, when sample vessels 15 containdifferent fermentation media 20 or undergo different fermentationconditions, numerous sensors 790 are optionally employed.

[0139] The above automated process is optionally used in conjunctionwith any fermentor apparatus or method to known to those of skill in theart. In particular, it is useful when practicing fermentation using thefermentors presented herein. However, it is noted that the examplespresented herein are provided for purposes of illustration and not oflimitation. While the foregoing invention has been described in somedetail for purposes of clarity and understanding, it will be clear toone skilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. For example, all the techniques and apparatusdescribed above may be used in various combinations and other uses forthe present invention are also contemplated. It is also noted thatequivalents for the particular embodiments discussed in this descriptionmay be used in the invention as well.

[0140] All publications, patents, patent applications, or otherdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, or other document wereindividually indicated to be incorporated by reference for all purposes.

What is claimed is:
 1. A fermentation apparatus comprising: (a) a container frame configured to contain a plurality of sample vessels; and, (b) a gas distribution arrangement that is configured to provide gas to a plurality of sample vessels when the sample vessels are positioned in the container frame.
 2. The fermentation apparatus of claim 1, wherein the gas distribution arrangement comprises a gas inlet configured to deliver gas to a plurality of cannulas, which cannulas are configured to provide gas to the sample vessels when the sample vessels are positioned in the container frame.
 3. The fermentation apparatus of claim 1, wherein the gas distribution arrangement comprises: (a) a dispensing plate that comprises a top portion and a bottom portion, wherein the bottom portion and the top portion are joined together such that a hollow space exists between the top portion and the bottom portion; (b) an array of sample vessel areas located in a bottom surface of the bottom portion, which sample vessel areas each comprise a recess and are positioned to correspond to an array of sample vessels; (c) an array of cannulas that are in fluid communication with the hollow space and protrude from a bottom surface of the dispensing plate through the sample vessel areas; and (d) a gas inlet in fluid communication with the hollow space for delivering gas into a plurality of sample vessels via the cannulas during fermentation.
 4. The fermentation apparatus of claim 3, wherein each of the cannulas comprises a plurality of passages.
 5. The fermentation apparatus of claim 4, wherein each of the cannulas comprises at least three passages.
 6. The fermentation apparatus of claim 3, wherein the gas distribution arrangement is configured to allow delivery of one or more reagent to the sample vessels.
 7. The fermentation apparatus of claim 1, wherein the container frame is configured to contain an array of sample vessels.
 8. The fermentation apparatus of claim 7, wherein the container frame is configured to contain an 8 by 12 array of sample vessels.
 9. The fermentation apparatus of claim 7, wherein the container frame is configured to contain at least 96 sample vessels.
 10. The fermentation apparatus of claim 9, wherein the container frame is configured to contain 96, 384, or 1536 sample vessels.
 11. The fermentation apparatus of claim 1, wherein the container frame is transportable.
 12. The fermentation apparatus of claim 11, wherein the container frame is configured for transport to a post-fermentation processing station.
 13. The fermentation apparatus of claim 1, wherein the container frame is configured for placement within a temperature controlled area, wherein a temperature controller is coupled to the container frame and/or to the plurality of sample vessels.
 14. The fermentation apparatus of claim 13, wherein the temperature controlled area comprises a water bath or a temperature controlled room.
 15. The fermentation apparatus of claim 1, wherein the container frame is autoclavable.
 16. The fermentation apparatus of claim 1, wherein the container frame and the gas distribution arrangement are autoclavable.
 17. The fermentation apparatus of claim 1, further comprising a plurality of sample vessels.
 18. The fermentation apparatus of claim 17, wherein each of the sample vessels has a volume of 50 to 100 ml.
 19. The fermentation apparatus of claim 17, wherein each sample vessel comprises a sample.
 20. The fermentation apparatus of claim 19, wherein each sample is 80 mls or less.
 21. The fermentation apparatus of claim 19, wherein the samples each have substantially the same composition.
 22. The fermentation apparatus of claim 19, wherein the samples each have a different composition.
 23. The fermentation apparatus of claim 17, wherein the sample vessels comprise glass, plastic, metal, polycarbonate, and/or ceramic.
 24. The fermentation apparatus of claim 17, wherein one or more of the sample vessels comprises a vent.
 25. The fermentation apparatus of claim 17, further comprising a sensor in contact with one or more of the samples in the sample vessels.
 26. The fermentation apparatus of claim 1, wherein the gas distribution arrangement comprises a gas source which gas source provides oxygen or a mixture of oxygen and at least one other gas to each sample vessel during operation of the apparatus.
 27. The fermentation apparatus of claim 1, further comprising a process controller operably coupled to the gas distribution arrangement.
 28. The fermentation apparatus of claim 1, further comprising a dispenser for dispensing one or more reagents into the plurality of sample vessels.
 29. The fermentation apparatus of claim 28, wherein the dispenser is configured to dispense the reagents into the plurality of sample vessels via a plurality of apertures that correspond to the sample vessels.
 30. A fermentor head for multiple sample fermentation, the fermentor head comprising: (a) a dispensing plate that comprises a top portion and a bottom portion, wherein the bottom portion and the top portion are joined together such that a hollow space exists between the top portion and the bottom portion; (b) an array of sample vessel areas located in a bottom surface of the bottom portion, which sample vessel areas each comprise a recess and are positioned to correspond to an array of sample vessels; (c) an array of cannulas that are in fluid communication with the hollow space and protrude from a bottom surface of the dispensing plate through the sample vessel areas; and (e) a gas inlet in fluid communication with the hollow space for delivering gas into a plurality of sample vessels via the cannulas during fermentation.
 31. The fermentor head of claim 30, wherein the dispensing plate further comprises an array of apertures for accessing samples during fermentation.
 32. The fermentor head of claim. 30, wherein the array of cannulas comprises an 8 by 12 array.
 33. The fermentor head of claim 30, wherein the array of cannulas comprises at least 96 cannulas.
 34. The fermentor head of claim 33, wherein the array of cannulas comprises 96, 384, or 1536 cannulas.
 35. The fermentor head of claim 30, wherein the cannulas extend 15 to 16 centimeters below the bottom surface of the first plate.
 36. The fermentor head of claim 30, wherein the sample vessels have a volume of 50 to 200 ml.
 37. The fermentor head of claim 30, wherein the sample vessels have a volume of 50 to 100 ml.
 38. The fermentor head of claim 30, wherein the cannulas deliver gas adjacent to a bottom of the sample vessels.
 39. The fermentor head of claim 30, wherein the gas inlet delivers oxygen or nitrogen into the interior space of the second plate, thereby providing oxygen or nitrogen to the sample vessels via the cannulas during fermentation.
 40. The fermentor head of claim 30, wherein each of the cannula comprises at least three passages.
 41. The fermentor head of claim 30, wherein the cannulas are adapted to deliver gas, deliver fluid, or aspirate fluid from the sample vessels during fermentation.
 42. A method of fermenting a plurality of samples, the method comprising: (a) providing a plurality of sample vessels in a container frame, wherein each of the sample vessels contains a sample; (b) fermenting the samples in the plurality of sample vessels, which fermenting comprises simultaneously delivering gas to each of the sample vessels via a plurality of cannulas associated with the sample vessels.
 43. The method of claim 42, wherein each sample has a volume of less than 100 ml.
 44. The method of claim 42, further comprising pre-processing or post-processing the samples in the sample vessels.
 45. The method of claim 44, wherein the pre-processing or post-processing is performed in a different location than step (b).
 46. The method according to claim 44, wherein the pre-processing and/or post-processing are performed robotically.
 47. The method according to claim 44, wherein the pre-processing and/or post-processing comprises centrifugation, aspiration, or dispensing of one or more reagent.
 48. The method of claim 42, wherein delivering gas comprises delivering oxygen, air, and/or, nitrogen to the samples.
 49. The method of claim 42, wherein delivering gas comprises delivering air and oxygen to the samples over a period of time, during which period of time, the ratio of air to oxygen changes.
 50. The method of claim 49, wherein the ratio changes linearly over time or in a stepwise manner over time.
 51. The method of claim 42, further comprising configuring the sample vessels into a rectangular array, a honeycomb array, or a linear array within the container frame.
 52. The method of claim 42, further comprising transferring the sample vessels into a centrifuge rotor.
 53. The method according to claim 42, further comprising detecting one or more fermentation conditions with a sensor coupled to one or more sample vessels and adjusting the fermentation conditions in the sample vessels.
 54. The method according to claim 53, comprising detecting and adjusting at pre-determined time intervals.
 55. The method according to claim 53, wherein the adjusting the fermentation conditions comprises adding a feed solution to the sample vessels.
 56. The method according to claim 53, wherein the detecting comprises: measuring a pH of one of the samples; measuring a redox potential of one of the samples; measuring an optical density of one of the samples; and/or measuring a light emission from one of the samples.
 57. The method of claim 42, further comprising autoclaving the sample vessels in the container frame.
 58. The method of claim 57, further comprising autoclaving the plurality of cannulas simultaneously with the sample vessels in the container frame.
 59. A method of fermenting a plurality of samples, the method comprising: (a) positioning a plurality of sample vessels into a transportable container frame, which container frame maintains the sample vessels in an array; (b) placing the plurality of samples into the plurality of sample vessels; (c) attaching a fermentor head to the container frame, which fermentor head comprises an array of cannulas, wherein the array of cannulas corresponds to the array of sample vessels and is inserted into the sample vessels; (d) fermenting the samples in the sample vessels, which fermenting comprising simultaneously delivering a gas to the samples via the array of cannulas.
 60. The method of claim 59, wherein step (c) is performed prior to step (b).
 61. The method of claim 59, wherein step (b) is performed prior to step (a).
 62. The method of claim 59, wherein delivering a gas comprising delivering oxygen, nitrogen, and/or air to the sample vessels during step (d).
 63. The method of claim 59, wherein step (d) is an anaerobic fermentation comprising delivering an inert gas to maintain anaerobic fermentation conditions in the sample vessels.
 64. The method of claim 59, wherein the sample vessels each have a volume between 50 and 200 ml.
 65. The method of claim 59, wherein the sample vessels have a volume between 80 and 100 ml.
 66. The method of claim 59, wherein each sample has a volume less than 200 ml.
 67. The method of claim 59, wherein each sample has a volume of less than 100 ml.
 68. The method of claim 69, comprising robotically transporting the sample vessels in the container frame.
 69. The method of claim 59, further comprising simultaneously transporting the plurality of sample vessels in the container frame to a processing station.
 70. The method of claim 69, wherein the processing station comprises a centrifuge, an aspirator, and/or a dispenser.
 71. The method of claim 70, wherein the sample container is compatible with the centrifuge.
 72. The method of claim 70, wherein the sample vessels are compatible with the centrifuge.
 73. The method of claim 70, further comprising removing the sample vessels from the container frame and introducing the sample vessels into the centrifuge.
 74. The method of claim 70, wherein the aspirator comprises an aspirator head which corresponds to the array of sample vessels within the container frame, the method further including operably attaching the aspirator head to the sample vessels and simultaneously aspirating the samples within the sample vessels.
 75. The method of claim 70, the method further dispensing one or more materials into the sample vessels.
 76. The method of claim 70, wherein the dispenser comprises a dispensing head corresponding to the array of sample vessels, the method further including operably attaching the dispenser head to the sample vessels and simultaneously dispensing one or more materials into the sample vessels.
 77. The method of claim 59, wherein the array comprises an 8 by 12 array.
 78. The method of claim 59, wherein the array comprises 96, 384, or 1536 sample vessels.
 79. The method of claim 59, further comprising positioning the sample vessels in the container frame in a water bath during the fermenting step in order to control the fermentation temperature.
 80. A method of processing a plurality of fermentation samples, the method comprising: (a) fermenting a plurality of fermentation samples in a plurality of sample vessels, resulting in a plurality of fermented samples; (b) robotically transporting the sample vessels containing the fermented samples to a centrifuge head; and (c) centrifuging the fermented samples in the same sample vessels in which the fermentation was performed.
 81. The method of claim 80, the method further including isolating a supernatant from the sample vessels after centrifuging the fermentation samples.
 82. The method of claim 80, wherein at least 4 sample vessels are robotically transported to the centrifuge head at the same time.
 83. The method of claim 80, wherein at least 10 sample vessels are robotically transported to the centrifuge head at the same time.
 84. The method of claim 80, wherein each sample vessel contains less than 100 mL of fermentation sample.
 85. The method of claim 80, wherein the plurality of sample vessels are held in an 8 by 12 array. 